Solvent effects on substrate and phosphate interactions with the (Na+ + K+)-ATPase

(Na+ + K+)-ATPase activity of a dog kidney enzyme preparation was markedly inhibited by 10-30% (v/v) dimethyl sulfoxide (Me2SO) and ethylene glycol (Et(OH)2); moreover, Me2SO produced a pattern of uncompetitive inhibition toward ATP. However, K+-nitrophenylphosphatase activity was stimulated by 10-20% Me2SO and Et(OH)2 but was inhibited by 30-50%. Me2SO decreased the Km for this substrate but had little effect on the Vmax below 30% (at which concentration Vmax was then reduced). Me2SO also reduced the Ki for Pi and acetyl phosphate as competitors toward nitrophenyl phosphate but increased the Ki for ATP, CTP and 2-O-methylfluorescein phosphate as competitors. Me2SO inhibited K+-acetylphosphatase activity, although it also reduced the Km for that substrate. Finally, Me2SO increased the rate of enzyme inactivation by fluoride and beryllium. These observations are interpreted in terms of the E1P to E2P transition of the reaction sequence being associated with an increased hydrophobicity of the active site, and of Me2SO mimicking such effects by decreasing water activity: (i) primarily to stabilize the covalent E2P intermediate, through differential solvation of reactants and products, and thereby inhibiting the (Na+ + K+)-ATPase reaction and acting as a dead-end inhibitor to produce the pattern of uncompetitive inhibition; inhibiting the K+-acetylphosphatase reaction that also passes through an E2P intermediate; but not inhibiting (at lower Me2SO concentrations) the K+-nitrophenylphosphatase reaction that does not pass through such an intermediate; and (ii) secondarily to favor partitioning of Pi and non-nucleotide phosphates into the hydrophobic active site, thereby decreasing the Km for nitrophenyl phosphate and acetyl phosphate, the Ki for Pi and acetyl phosphate in the K+-nitrophenylphosphatase reaction, accelerating inactivation by fluoride and beryllium acting as phosphate analogs, and, at higher concentrations, inhibiting the K+-nitrophenylphosphatase reaction by stabilizing the non-covalent E2.P intermediate of that reaction. In addition, Me2SO may decrease binding at the adenine pocket of the low-affinity substrate site, represented as an increased Ki for ATP, CTP and 3-O-methylfluorescein phosphate.     

Kinetics and thermodynamics of catalysis by the inorganic pyrophosphatase of Escherichia coli in both directions

Combined evidence obtained from the measurements of pyrophosphate hydrolysis and synthesis, oxygen exchange between phosphate and water, enzyme-bound pyrophosphate formation and Mg2+ binding enabled us to deduce the overall scheme of catalysis by Escherichia coli inorganic pyrophosphatase in the presence of Mg2+. We determined the equilibrium constants for Mg2+ binding to various enzyme species and forward and reverse rate constants for the four steps of the catalytic reaction, namely, binding/release of PPi, hydrolysis/synthesis of PPi and successive binding/release of two Pi molecules. Catalysis by the E. coli enzyme in both directions, in contrast to baker's yeast pyrophosphatase, occurs via a single pathway, which requires the binding of Mg2+ to the sites of four types. Three of them can be filled in the absence of the substrates, and the affinity of one of them to Mg2+ is increased by two orders of magnitude in the enzyme-substrate complexes. The distribution of 18O-labelled phosphate isotopomers during the exchange indicated that hydrolysis of pyrophosphate in the active site is appreciably reversible. The equilibrium constant for this process estimated from direct measurements is 5.0. The ratio of the maximal velocities of pyrophosphate hydrolysis and synthesis is 69. The rate of the synthesis is almost entirely determined by the rate of the release of pyrophosphate from the enzyme. In the hydrolytic reaction, enzyme-bound pyrophosphate hydrolysis and successive release of two phosphate molecules proceed with nearly equal rate constants.     

Stimulatory and inhibitory effects of dimethyl sulfoxide and ethylene glycol on ATPase activity and calcium transport of sarcoplasmic membranes.

1. The effect of dimethyl sulfoxide (Me2SO) and ethylene glycol on two different preparations of the sarcoplasmic reticulum, i.e. native membranes and membranes whose phospholipids were hydrolyzed by phospholipase A, were investigated using ATP and p-nitrophenylphosphate as substrates. 2. Me2SO and ethylene glycol inhibit both calcium-dependent ATP hydrolysis and ATP-supported calcium transport by native vesicles. 3. In contrast, calcium-dependent p-nitrophenylphosphatase activity as well as p-nitrophenyl-phosphate-supported calcium transport are activated by both agents at concentrations lower than 30% (v/v). 4. Me2SO strongly stimulates p-nitrophenylphosphate activity of vesicles treated with phospholipase A, but has relatively little effect on p-nitrophenylphosphatase activity of native vesicles. 5. Up to a concentration of approximately 40% Me2SO (v/v) the inhibiting effect on the calcium-dependent ATPase is fully reversible, but only partially reversible on calcium transport. 6. In the concentration range where Me2SO inhibits ATP hydrolysis and calcium transport, it does not affect ATP binding to the membranes nor calcium-dependent formation of phospho-protein. 7. The rate of dephosphorylation as well as the rate of Pi exchange between ATP and ADP are markedly reduced by the presence of 30% Me2SO (v/v). 8. While Me2SO inhibits passive calcium efflux, ethylene glycol produces a considerable activation. 9. ADP-dependent calcium efflux and ATP synthesis are activated by 15% Me2SO (v/v). Ethylene glycol reduces both activities. 10. The results suggest that the respective substrate-enzyme complexes are differently affected by the agents, resulting either in inhibition or stimulation     

The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.     

Enzyme mechanism and catalytic property of beta propeller phytase

BACKGROUND: Phytases hydrolyze phytic acid (myo-inositol-hexakisphosphate) to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are used in animal feed to reduce phosphate pollution in the environment. Recently, a thermostable, calcium-dependent Bacillus phytase was identified that represents the first example of the beta propeller fold exhibiting phosphatase activity. We sought to delineate the catalytic mechanism and property of this enzyme. RESULTS: The crystal structure of the enzyme in complex with inorganic phosphate reveals that two phosphates and four calcium ions are tightly bound at the active site. Mutation of the residues involved in the calcium chelation results in severe defects in the enzyme's activity. One phosphate ion, chelating all of the four calcium ions, is close to a water molecule bridging two of the bound calcium ions. Fluoride ion, which is expected to replace this water molecule, is an uncompetitive inhibitor of the enzyme. The enzyme is able to hydrolyze any of the six phosphate groups of phytate. CONCLUSIONS: The enzyme reaction is likely to proceed through a direct attack of the metal-bridging water molecule on the phosphorous atom of a substrate and the subsequent stabilization of the pentavalent transition state by the bound calcium ions. The enzyme has two phosphate binding sites, the "cleavage site", which is responsible for the hydrolysis of a substrate, and the "affinity site", which increases the binding affinity for substrates containing adjacent phosphate groups. The existence of the two nonequivalent phosphate binding sites explains the puzzling formation of the alternately dephosphorylated myo-inositol triphosphates from phytate and the hydrolysis of myo-inositol monophosphates.     

Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state "burst" of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.     

Effect of pressure and calcium on the reversible inhibition of the sarcoplasmic-reticulum calcium-transport enzyme and on its tryptic cleavage pattern

The reversible inhibition of the sarcoplasmic-reticulum calcium-transport enzyme by pressure at room temperature is accompanied by a significant enhancement of the accessibility of the enzyme to tryptic cleavage dependent on the presence of calcium. The calcium-transport enzyme activity was monitored with dinitrophenyl phosphate as substrate. Pressure in the range 0.1-100.0 MPa affects trypsin cleavage of the control substrate N-alpha-benzoyl-L-arginine-4-nitroanilide hydrochloride little in the presence and absence of calcium. In contrast, application of 100.0 MPa to the calcium-transport enzyme at room temperature accelerates subsequent tryptic cleavage at the T2 but not at the T1 cleavage site [C. J. Brandl et al. (1986) Cell 44, 597-607]. Pressure application during tryptic digestion likewise solely affects cleavage at T2 which proceeds slowly in the absence but rapidly in the presence of calcium. At atmospheric pressure in the absence of calcium and at high pressure in the absence and presence of calcium new cleavage sites are exposed giving rise to new subfragments B1-3 in addition to the established peptides A1 and A2. Under pressure and in the presence of calcium, A1 and A2 rapidly disappear indicating the presence of calcium-binding sites in these peptides. In contrast, the B1-3 peptides which are most likely derivates of the B fragment accumulate in the presence and absence of calcium. In contrast to tryptic cleavage at atmospheric pressure, tryptic cleavage of the A as well as the B fragment tends to completion under pressure. In parallel to the disappearance of the A and B fragments calcium-dependent substrate hydrolysis vanishes. Computation of activation volumes for pressure-induced reversible enzyme inhibition and for tryptic cleavage furnished closely related volumes of opposite signs of 20-40 ml/mol and 80-100 ml/mol in the ranges 0.1-40.0 MPa and 40.0-100.0 MPa, respectively. Thus pressure produces reversible changes in the calcium-transport enzyme which activates and modifies tryptic-cleavage patterns at the T2 site of the A segment and at sites in its subfragments in the presence of calcium, i.e. if the enzyme residues in its E1 state. In contrast tryptic cleavage of the B fragment is accelerated by pressure independently of the presence of calcium.     

Biological and pathological functions of phospholipase A(2) receptor

In 40% dimethyl sulfoxide (Me2SO) high-affinity ouabain (O) binding to Na,K-ATPase (E) is promoted by Mg2+ in the absence of inorganic phosphate (Pi) (Fontes et al., Biochim. Biophys. Acta 1104, 215-225, 1995). Furthermore, in Me2SO the EO complex reacts very slowly with Pi and this ouabain binding can therefore be measured by the degree of inhibition of rapid phosphoenzyme formation. Here we found that, unexpectedly, the ouabain binding decreased with the enzyme concentration in the Me2SO assay medium. We extracted the enzyme preparation with Me2SO or chloroform/methanol and demonstrated that the extracted (depleted) enzyme bound ouabain poorly. Addition of such extracts to assays with low enzyme concentration or depleted enzyme fully restored the high-affinity ouabain binding. Dialysis experiments indicated that the active principle had a molecular mass between 3.5 and 12 kDa. It was highly resistant to proteolysis. It was suggested that the active principle could either be a low-molecular-weight, proteolysis-resistant-peptide (e.g., a proteolipid) or a factor with a nonproteinaceous nature. A polyclonal antibody raised against the C-terminal 10 amino acids of the rat kidney gamma-subunit was able to recognize this low-molecular-weight peptide present in the extracts. The previously depleted enzyme displayed lower amounts of the gamma-proteolipid in comparison to the native untreated enzyme, as demonstrated by immunoreaction with the antibody.     

Modification of ligand binding to the Na+/K+-activated ATPase

Interactions between the ligands Mg2+, K+, and substrate and the Na+/K+-activated ATPase were examined in terms of a rapid-equilibrium, random-order, terreactant kinetic scheme for the K+-nitrophenyl phosphatase reaction that is catalyzed by this enzyme. At 37 degrees C and pH 7.5 the derived values for the dissociation constants from the free enzyme were 0.2, 0.08, and 1.4 mM for Mg2+, K+, and substrate, respectively. For Mg2+ interactions, the presence of 20% (v/v) dimethyl sulfoxide (Me2SO) increased the calculated affinity 25-fold; higher concentrations increased affinity still further. Neither reducing the temperature to 20 degrees C nor altering the pH from 6.5 to 8.3 appreciably changed the affinity for Mg2+ in the absence or presence of Me2SO. The Mg2+ sites are thus characterized by an absence of functional groups ionizable in the pH range 6.5-8.3, with binding driven by entropy changes, and with Me2SO, probably through solvation effects on the protein, increasing affinity for Mg2+ close to that for Ca2+ and Mn2+. By contrast, for K+ interactions, the presence of 20% Me2SO increased the calculated affinity only by half; moreover, reducing the temperature to 20 degrees C and the pH to 6.5 both increased affinity and diminished the response to Me2SO. The K+ sites are thus characterized by a marked sensitivity to pH and temperature, presumably through alterations in enzyme conformational equilibria that in turn are modifiable by Me2SO. Inhibition by higher concentrations of Mg2+, which varies inversely with the K+ concentration, was decreased by Me2SO. Finally, for substrate interactions, the presence of 20% Me2SO increased the calculated affinity 4-fold, and, as for Mg2+-binding, neither reducing the temperature nor varying the pH over the range 6.5-8.3 appreciably altered the affinity in the absence or presence of Me2SO. Thus, the substrate sites, like the Mg2+ sites, are characterized by an absence of functional groups ionizable in this range, with binding driven by entropy changes, and with Me2SO increasing affinity for substrate, in this case probably through favoring the partitioning of substrate from the medium into the hydrophobic active site.     

Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage

Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.     

Effects of pressure on enzyme function of Escherichia coli dihydrofolate reductase

To elucidate the effects of pressure on the function of Escherichia coli dihydrofolate reductase (DHFR), the enzyme activity and the dissociation constants of substrates and cofactors were measured at pressures up to 250 MPa at 25 degrees C and pH 7.0. The enzyme activity decreased with increasing pressure, accompanying the activation volume of 7.8 ml mol(-1). The values of the Michaelis constant (K(m)) for dihydrofolate and NADPH were slightly higher at 200 MPa than at atmospheric pressure. The hydride-transfer step was insensitive to pressure, as monitored by the effects of the deuterium isotope of NADPH on the reaction velocity. The dissociation constants of substrates and cofactors increased with pressure, producing volume reductions from 6.5 ml mol(-1) (tetrahydrofolate) to 33.5 ml mol(-1) (NADPH). However, the changes in Gibbs free energy with dissociation of many ligands showed different pressure dependences below and above 50 MPa, suggesting conformational changes of the enzyme at high pressure. The enzyme function at high pressure is discussed based on the volume levels of the intermediates and the candidates for the rate-limiting process.     

The initial phosphate burst in ATP hydrolysis by myosin and subfragment-1 as studied by a modified malachite green method for determination of inorganic phosphate

The Malachite Green method for determination of inorganic phosphate (Pi) (Itaya K. & Ui, M. (1966) Clin. Chim. Acta 14, 361-366) was modified to measure Pi in the range of 0.2-15 nmol per ml of ATPase reaction mixture. An ATPase reaction mixture is quenched with an equal volume of 0.6 M PCA; the supernatant after centrifugation is mixed with an equal volume of the Malachite Green/molybdate reagent containing 2 g of sodium molybdate, 0.3 g of Malachite Green and 0.5 g of Triton X-100 or Sterox SE in 1 liter of 0.7 M HCl, and the absorbance at 650 nm is then measured after a 35-40 min incubation at 25 degrees C. Owing to the high sensitivity and simplicity of the modified method, the slow time course of myosin ATP hydrolysis in the presence of Mg2+ and the size of initial phosphate burst can be determined accurately using relatively low concentrations of native myosin and its subfragment-1. The phosphate burst size varied with changes in pH, ionic strength, and temperature. A typical value was 0.8-0.9 mol per site in 0.1 M KCl, 10 mM MgCl2, pH 8.0 at 25 degrees C for fresh enzyme preparations.     

Fundamental cryobiology of rat immature and mature oocytes: hydraulic conductivity in the presence of Me(2)SO, Me(2)SO permeability, and their activation energies

The hydraulic conductivity in the presence of dimethyl sulfoxide Me(2)SO (L(p)(Me(2)SO)), Me(2)SO (P(Me(2)SO)) permeability and reflection coefficient (sigma) of immature (germinal vesicle; GV) and mature (metaphase II; MII) rat oocytes were determined at various temperatures. A temperature controlled micropipette perfusion technique was used to conduct experiments at five different temperatures (30, 20, 10, 4, and -3 degrees C). Kedem and Katchalsky membrane transport theory was used to describe the cell volume kinetics. The cell volumetric changes of oocytes were calculated from the measurement of two oocyte diameters, assuming a spherical shape. The activation energies (E(a)) of L(p)(Me(2)SO) and P(Me(2)SO) were calculated using the Arrhenius equation. Activation energies of L(p)(Me(2)SO) for GV and MII oocytes were 34.30 Kcal/mol and 16.29 Kcal/mol, respectively; while the corresponding E(a)s of P(Me(2)SO) were 19.87 Kcal/mol and 21.85 Kcal/mol, respectively. These permeability parameters were then used to calculate cell water loss in rat oocytes during cooling at subzero temperatures. Based on these values, the predicted optimal cooling rate required to maintain extra- and intracellular water in near equilibrium for rat GV stage oocytes was found to be between 0.05 degrees C/min and 0. 025; while for rat MII oocytes, the corresponding cooling rate was 1 degrees C/min. These data suggest that standard cooling rates used for mouse oocytes (e.g., 0.5-1 degrees C/min) can also be employed to cryopreserve rat MII oocytes. However, the corresponding cooling rate required to avoid damage must be significantly slower for the GV stage rat oocyte. J. Exp. Zool. 286:523-533, 2000.     

Dimethylsulfoxide oxidizes glutathione in vitro and in human erythrocytes: kinetic analysis by 1H NMR

The interaction of dimethylsulfoxide (Me2SO) with glutathione was investigated under non-equilibrium conditions in solution using 1H NMR and in intact erythrocytes using 1H spin-echo NMR. In solution the reaction was observed to follow second-order kinetics (Rate = k1[glutathione][Me2SO]) at 300 K pH 7.4, k(sol) = 4.7 x 10(-5)mol(-1)L(1)s(-1). In intact erythrocytes the rate constant for the cellular environment, k(cell), was found to be slightly larger at 8.1 x 10(-5)mol(-1)L(1)s(-1). Furthermore, the reaction of Me2SO with erythrocyte glutathione showed a biphasic dependence on the Me2SO concentration, with little oxidation of glutathione occurring until the Me2SO concentration exceeded 0.5 molL(-1). The results suggest that at lower concentrations, Me2SO can be effectively removed, most probably by reaction with glutathione, which is regenerated by glutathione reductase, although preferential reaction with other cellular components (e.g., membrane or cellular thiols) cannot be ruled out. Thus the concentrations of Me2SO that are commonly used in cryopreservation of mammalian cells ( approximately 1.4 molL(-1)) can cause oxidation of intracellular glutathione.     

Stopped-flow fluorescence and steady-state kinetic studies of ligand-binding reactions of glucoamylase from Aspergillus niger.

The presteady-state and steady-state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition-state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial-rate, stopped-flow and steady-state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three-step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non-enzymic hydrolysis the transition-state stabilization energy of glucoamylase is approximately -66 kJ/mol with maltose and only -14 kJ/mol with isomaltose. Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.     

High-pressure investigations of cytochrome P-450 spin and substrate binding equilibria

The effects of high pressure (1-2000 bar) on the spin state and substrate binding equilibria in cytochrome P-450 have been determined. The high-spin (S = 5/2) to low spin (S = 1/2) transition of the ferric hemoprotein was monitored by uv-visible spectroscopy at various substrate concentrations. Increasing hydrostatic pressure on a sample of substrate-bound cytochrome P-450 resulted in a decrease in the high-spin fraction as monitored by a Soret maxima at 391 nm and an increase in the low-spin 417-nm region of the spectrum. These pressure-induced optical changes were totally reversible for all pressures below 800 bar and were found to correspond to simple substrate dissociation from the enzyme. High levels of the normally metabolized substrate, d-camphor, corresponding to a 99.9% saturation of the hemoprotein active site (50 mM Tris-Cl, 100 mM KCl, pH 7.2) completely prevented the pressure-induced high-spin to low-spin transition that is observed at less than saturating substrate concentrations. A gradual increase in the formation of the inactive P-420 form of the cytochrome was noted if the pressure of the sample was increased above 800 bar. These pressure-linked spectral changes were used to determine the microscopic volume change accompanying substrate binding, which was found to be -47.0 +/- 2 ml/mol (pH 7.2) which represents a substantial change for a ligand dissociation reaction. The observed volume change for camphor binding decreases to -30.6 +/- 2 ml/mol at pH 6.0, suggesting the involvement of a linked proton equilibrium. Various substrate analogs of camphor induce varying degrees of low-spin to high-spin shift upon binding to ferric cytochrome P-450 (3). The volume changes for the dissociation of these substrates were very similar to those obtained with camphor. The conformational changes associated with a shift from high- to low-spin ferric iron appear to be small in comparison to the overall macroscopic changes in volume accompanying substrate binding to the enzyme.     

FTIR spectroscopic kinetic analysis of alkaline phosphatase under hyperbaric manipulation.

For the first time, high-pressure infrared spectroscopy has been used in an enzyme kinetics study. This technique allows not only the investigation of kinetics under very high pressure, but it also allows simultaneous determinion of changes in the secondary structure of enzymes at the corresponding pressures. In the present study, a classical enzyme reaction, the conversion of p-nitrophenol phosphate into p-nitrophenol by alkaline phosphatase was selected to demonstrate the potential of infrared spectroscopy as an alternative physical method in the high-pressure study of enzyme kinetics. The rate constants of this enzyme reaction have been determined as a function of pressure in the pressure range 0.001-14 kbar. The first-order rate constants thus obtained increases with increasing pressure up to 8.3 kbar. At this pressure, the reaction rate decreases abruptly due to the denaturation of the enzyme arising from the conformational changes of some alpha-helical segments in the enzyme molecules into beta-sheet structure. The present results suggest that the pressure-enhanced overall hydrogen-bond strength in the amide groups of the enzyme is one of the factors which stimulate the enzyme activity. Moreover, the dissociation of the dimeric enzyme into its subunits does not inhibit the enzyme activity but only attributes to a slight change in activation volume.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (2H2O) and dimethyl sulfoxide (Me2SO) on [3H]ouabain binding to (Na+,K+)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg2+ + ATP + Na+). In contrast, both solvents stimulated type II (i.e., Mg2+ + Pi-, Mg2+-, or Mn2+-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg2+ + Na+ + ATP, 75% in the Mg2+ + Pi system, and in the presence of Mn2+ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus 2H2O inhibition of type I ouabain binding was dependent on Na+ concentration in the reaction and was reduced as Na+ was elevated. Contact of the enzyme with the Me2SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na+ + ATP first and then to Me2SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me2SO and 2H2O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H2O, and E1 enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H2O at the active enzyme center and E2 enzyme conformation. This postulation of a role of H2O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.     

Calorimetric determination of thermodynamic parameters of reaction reveals different enthalpic compensations of the yeast hexokinase isozymes

The change in enthalpy and rate constants for the reactions of yeast hexokinase isozymes, PI (Hxk1) and PII (Hxk2), was determined at pH 7.6 and 25 degrees C by isothermal titration calorimetry. The reactions were done in five buffer systems with enthalpy of protonation varying from -1.22 kcal/mol (phosphate) to -11.51 kcal/mol (Tris), allowing the determination of the number of protons released during glucose phosphorylation. The reaction is exothermic for both isozymes with a small, but significant (p < 0.0001), difference in the enthalpy of reaction (Delta HR), with an Delta HR of -5.1 +/- 0.2 (mean +/- S.D.) kcal/mol for Hxk1, and an Delta HR of -3.3 +/- 0.3 (mean +/- S.D.) kcal/mol for Hxk2. The Km for ATP determined by ITC was very similar to those reported in the literature for both isozymes. The effect of NaCl and KCl, from 0 to 200 mM, showed that although the rate of reaction decreases with increasing ionic strength, no change in the Delta HR was observed suggesting an entropic nature for the ionic strength. The differences in Delta HR obtained here for both isozymes strongly suggest that, besides glucose phosphorylation, another side reaction such as ATP hydrolysis and/or enzyme phosphorylation is taking place.